1. Field of the Invention
The present invention relates generally to methods for assessing human female ovarian health by evaluating CGG repeats on the fragile X mental retardation 1 (FMR1) gene, and providing testing and treatment based on the evaluation. Particularly, the present invention provides methods for early detection of risk of infertility and/or imminent premature ovarian aging in a human female who has not experienced infertility and is not otherwise indicated to have premature ovarian aging. The present invention also provides methods for treatment of imminent premature ovarian aging in a human female, predicting infertility and determining the imminence of premature ovarian aging by analyzing the FMR1 gene and then performing a treatment and/or testing regimen depending on the results of the analysis. No prior known tests can detect imminent ovarian aging or infertility in a human female who has not experienced infertility and is not otherwise indicated to have premature ovarian aging.
2. Description of the Related Art
The following acronyms are used throughout this specification:
AIRE: Autoimmune Regulator
CGG: Cytosine-Guanine-Guanine
FMR1: Fragile X Mental Retardation 1
FMRP: Fragile X Mental Retardation Protein
FOR: Functional Ovarian Reserve
FXS: Fragile X Syndrome
OPOI: Occult Primary Ovarian Insufficiency
OR: Ovarian Reserve
POA: Premature Ovarian Aging
POF: Premature Ovarian Failure
POI: Primary Ovarian Insufficiency
POS: Premature Ovarian Senescence
TOR: Total Ovarian Reserve
These acronyms also appear after the first use of each full term.
The FMR1 gene (gene location Xq27.3) is commonly studied or analyzed because of its association with Fragile X syndrome (FXS). FXS is the most common cause of familial mental retardation and autism (see, Bagni C., Tassone F., Neri G., Hagerman R., Fragile X Syndrome: Causes, Diagnosis, Mechanisms, and Therapeutics, The Journal of Clinical Investigation, December 2012, 4314-22, hereinafter referred to as “Bagni”). FXS occurs when the FMR1 gene is inactivated and does not produce Fragile X Mental Retardation Protein (FMRP). FMRP is important for proper neurological development and is involved in RNA translation. This inactivation is usually caused by too many Cytosine-Guanine-Guanine (CGG) trinucleotide repeats on the FMR1 gene. FMR1 genes are usually classified by the number of such CGG repeats on the gene. The usual classification in current medical practice recognizes four ranges of CGG repeats on the FMR1 gene: a normal (or common) range of CGGn<45, an intermediate range of CGGn˜45-54, a premutation range of CGGn˜55-200 and a full mutation range of CGGn>200. FXS usually occurs in persons with an FMR1 gene in the full mutation range. A gene in the premutation range can expand to the full mutation range in the next generation of offspring (see, Willemsen R., Levenga J., Oostra B. A., CGG Repeat in the FMR1 Gene: Size Matters, Clinical Genetics, September 2011; 214-25, hereinafter referred to as “Willemsen”). Because of such expansion, FXS risk screening focuses on women with FMR1 genes in the premutation range, who are at risk for having children with FXS. FXS risk screening is the primary purpose of FMR1 testing in current medical practice.
Y. H. Fu found a peak in the population distribution of CGG repeats in the range CGGn=29-30 (see, Fu Y. H., Kuhl D. P., Pizzuti A., et al., Variation of the CGG Repeat at the Fragile X Site Results in Genetic Instability: Resolution of the Sherman Paradox, Cell, December 1991; 1047-58, hereinafter referred to as “Fu”). The inventors herein investigated a connection between the FMR1 gene and ovarian function based on the distribution peak at CGGn=29-30. Ovarian effects of the FMR1 gene are supported by a known association between FMR1 genotypes in the premutation range (CGGn˜55-200) and primary ovarian insufficiency (POI), also known as premature ovarian failure (POF) (see, Gleicher N., Weghofer A., Barad D. H., Defining Ovarian Reserve to Better Understand Ovarian Aging. Reproductive Biology and Endocrinology, February 2011, 23, hereinafter referred to as “Gleicher I”). A recent study of a mouse FMR1 homologue also supports the association of the FMR1 gene with ovarian aging (see, Hoffman G. E., Le W. W., Entezam A., et al. Ovarian Abnormalities in a Mouse Model of Fragile X Primary Ovarian Insufficiency. The Journal of Histochemistry and Cytochemistry, June 2012, 439-56).
Based on their research, the inventors defined new ranges of CGG repeats on the FMR1 gene relevant to ovarian health: a normal (norm) range of CGGn=26-34, a low range of CGGn<26 and a high range of CGGn>34. Further refinement of these ranges defined norm (both alleles in normal range), heterozygous (het, one allele in and the other outside normal range) and homozygous (hom, both alleles outside normal range) genotypes. Het and hom genotypes were further subdivided into high or low. For example, a female with a het-high genotype has one FMR1 allele with more than 34 CGG repeats and one FMR1 allele with between 26-34 CGG repeats. Cross-sectional studies demonstrate associations between the various genotypes described above and specific ovarian aging patterns (see, Gleicher N., Weghofer A., Barad D. H., Ovarian reserve Determinations Suggest New Function of FMR1 (Fragile X Gene) in Regulating Ovarian Ageing. Reproductive Biomedicine Online, June 2010, 768-75, hereinafter referred to as “Gleicher II”; Gleicher N., Weghofer A., Lee I. H., Barad D. H., FMR1 Genotype With Autoimmunity-Associated Polycystic Ovary-Like Phenotype and Decreased Pregnancy Chance. PloS One, December 2010, e15303, hereinafter referred to as “Gleicher III”; Gleicher N., Weghofer A., Lee I. H., Barad D. H., Association of FMR1 Genotypes With in Vitro Fertilization (IVF) Outcomes Based on Ethnicity/Race, PloS One, April 2011, e18781, hereinafter referred to as “Gleicher IV”; and Gleicher N., Weghofer A., Kim A., Barad D. H., The Impact in Older Women of Ovarian FMR1 Genotypes and Sub-Genotypes on Ovarian Reserve, PloS One, March 2012, e33638, hereinafter referred to as “Gleicher V”). These associations are more fully disclosed herein. Genotype/phenotype interactions are usually studied in homozygous subjects, but these studies have so far only studied norm and het women because all three hom sub-genotypes (high/high, high/low and low/low), combined, occur in less than 10 percent of women, not enough to fully study (see, Gleicher II, Gleicher III, Gleicher IV and Gleicher V). These new range and genotype definitions allow the use of the FMR1 gene to assess ovarian health.
Human females are typically tested to determine ovarian health and to assess their fertility only if they are experiencing infertility, at risk for infertility based on age and/or are indicated to have ovarian aging by showing signs of ovarian aging. These tests are for anti-Müllerian hormone (AMH) and/or follicle stimulating hormone (FSH) levels. Tests for FSH levels include tests for estradiol levels because a high estradiol level can suppress FSH levels. Such combined tests are referred herein to as FSH tests or FSH/estradiol tests. The tests are performed once and the human female's level of AMH and/or FSH is compared against the normal range for human females of her age. If AMH is lower than the normal range or FSH and/or estradiol is higher than normal the normal range, the human female is considered to have premature ovarian aging (POA), also known as occult premature ovarian insufficiency (OPOI). These AMH/FSH tests, however, are generally not performed in young human females, defined herein to mean human females who have not experienced infertility and are not otherwise indicated to have ovarian aging.
Because testing for ovarian health is presently performed only when the human female already experienced infertility and/or is indicated for ovarian aging by symptoms, such as menstrual irregularities, the diagnosis of POA or POF is usually only obtained when the POA is at advanced clinical stages, POF has occurred or the human female is about 38 years or older. As a result, there is an absence of prospective risk assessments in adolescent and young adult females even though approximately 10% of human females will suffer from premature ovarian aging. At advanced clinical stage or advanced age, even advanced fertility treatments for POA demonstrate only limited success, and egg donation remains the only realistic choice for women with POF (see, Gleicher I). Late diagnosis, of course, assumes further significance in older, often single women because POA further compounds the negative effects of advanced age. As a result, late diagnosis of POA leads to limited success in treatment.
Earlier diagnosis of premature ovarian aging presents many benefits for women, most notably, earlier and potentially more effective treatment options (see, Cil A. P., Bang H., Oktay K., Age-Specific Probability of Live Birth With Oocyte Preservation: An Individual Patient Data Meta-Analysis, Fertility and Sterility, August 2013, 492-9). Identification of human females likely to be affected by POA when their ovarian reserve (OR) is still relatively normal offers a choice between childbirth at a younger age than they otherwise planned or fertility preservation by assisted reproductive technologies. All methods of fertility preservation are more efficient at younger than older ages and, therefore, less costly and more cost-effective. The reduced cost is especially important given ever-increasing medical costs and the present high cost of infertility testing and treatment, which, in many cases, is not covered by health insurance.
Fertility preservation for young women is relatively recent and resulted from a need by women who became infertile after undergoing cancer treatment but who still desired to have children. Fertility preservation emerged to provide young cancer survivors a reproductive future (see, Waimev K. E., Duncan F. E., Su H. I., Smith K., Wallach H., Jona K., Coutifaris C., Gracia C. R., Shea L. D., Brannigan R. E., Chang R. J., Zelinski M. B., Stouffer R. L., Taylor R. I., Woodruff T. K., Future Directions in Oncofertility and Fertility Preservation: A Report From the 2011 Oncofertility Consortium Conference, Journal of Adolescent and Young Adult Oncology, March 2013, 25-30). Aside from fertility preservation for cancer patients, women are delaying childbirth for various social and personal reasons and use fertility preservation to have children later in life (see, Donnez J., Introduction: Fertility Preservation, from Cancer to Benign Disease to Social Reasons: The Challenge of the Present Decade, Fertility and Sterility, May 2013, 1467-1468; and Cobo A., Garcia-Velasco J. A., Domingo J., Remohl J., Pellicer A., Is Vitrification of Oocytes Useful for Fertility Preservation for Age-Related Fertility Decline and in Cancer Patients? Fertility and Sterility, May 2013, 1485-1495). Fertility preservation in response to causes of infertility other than cancer or voluntary delay, such as endometriosis, is also entering medical practice (see, Bedoschi G., Turan V., Oktay K., Fertility Preservation Options in Women with Endometriosis, Minerva Ginecologica, April 2013, 99-103). However, fertility preservation in response to other causes of infertility, such as premature ovarian aging, has not yet received attention because premature ovarian aging was not predictable by the existing knowledge in the art.
Ovarian aging is the combination of declines in oocyte quality and oocyte number. Ovulation, the maturation and release of oocytes, begins at menarche, the onset of menstrual cyclicity. Menarche is the start of a complex process of steady follicle recruitment that organizes recruited follicles into maturing monthly cohorts, groups of follicles in the same stage of development. In natural ovulation cycles, follicular cohorts mature over 2-4 months, resulting in ovulation of a single dominant follicle. The other follicles in the cohort undergo degeneration and apoptosis (see, FIG. 1), resulting in unifollicular ovulation. The ovary's ability to organize cohesive monthly cohorts of follicles of similar sizes and maturity is a characteristic of young age and normal ovarian function. The ability to organize and carry out monthly unifollicular ovulation diminishes with advancing female age and/or in association with POA (and possibly early stages of POF). Older females and patients with POA have more inhomogeneous follicle sizes and oocyte maturity distribution than females who are young and not experiencing POA. This difference is shown in IVF studies for those two populations (see, Gleicher I).
As FIG. 1 also shows, the current medical understanding holds that females are born with a limited pool of follicles, also known as the total ovarian reserve (TOR), that depletes throughout life until menopause. TOR peaks in intrauterine life at approximately 7 million follicles/oocytes, with significant depletion before birth. Females have less than 1 million follicles/oocytes at birth and by menarche approximately only 400,000 remain in the female. The speed of ovarian depletion slows between menarche and menopause, when only a few hundred to one thousand follicles/oocytes remain in the ovaries (see, Gleicher I).
A patient's TOR is primarily the large pool of unrecruited, primordial follicles “resting” at a very primitive stage. A patient's recruited follicles (also called “growing” follicles) are a much smaller part of TOR known as the functional ovarian reserve (FOR). After weeks to months of maturation, the recruited follicles reach maturity in either natural or ovarian stimulation cycles. A patient's TOR and FOR deplete over time and reflect the patient's ovarian age.
The genetic basis of follicle recruitment and its effect on TOR and FOR are not completely understood. The genes involved in follicle recruitment appear to limit over-recruitment of primordial follicles, which can rapidly deplete unrecruited follicles. When genes that affect follicle recruitment in either rodents or humans are mutated, blocked or knocked out, primordial follicles are over-recruited and deplete rapidly. Genes involved in follicle recruitment also influence a female's age at menopause. The primary function of these genes, therefore, appears to reduce the rate of follicular recruitment. Slower recruitment preserves more follicles/oocytes, leading to better remaining TOR at later ages.
The speed of follicle recruitment is statistically correlated to the number of remaining primordial follicles. Therefore, the size of the pool of growing follicles (representing FOR) also correlates with speed of recruitment (see, Gleicher V; Gleicher I; and Nelson S. M., Anderson R. A., Broekmans F. J., Raine-Fenning N., Fleming R., La Marca A., Anti-Müllerian Hormone: Clairvoyance or Crystal Clear? Human Reproduction, March 2012, 631-636, hereinafter referred to as “Nelson I”). AMH is produced in the granulosa cells of these small growing follicles and inhibits follicle recruitment and growth (see, Gleicher I; Ledger W. L., Clinical Utility of Measurement of Anti-Müllerian Hormone in Reproductive Endocrinology. Journal of Clinical Endocrinology & Metabolism, December 2010, 5144-5154, hereinafter referred to as “Ledger”; and Gleicher N., Weghofer A., Barad D. H., The Role of Androgens in Follicle Maturation and Ovulation Induction: Friend or Foe of Infertility Treatment? Reproductive Biology and Endocrinology, August 2011, 116). Because of this connection between AMH and the small growing follicles, a human female's AMH levels reflect the size of her pool of small growing follicles. Age-specific AMH levels, which reflect age-specific follicle pool size, are known in the art (see, Barad D. H., Weghofer A., Gleicher N., Utility of Age-Specific Serum Anti-Müllerian Hormone Concentrations, Reproductive Biomedicine Online, March 2011, 284-291, hereinafter referred to as “Barad”; and Kelsey T. W., Wright P., Nelson S. M., Anderson R. A., Wallace W. H. B., A Validated Model of Serum Anti-Müllerian Hormone from Conception to Menopause, PLoS One 2011, e22024, hereinafter referred to as “Kelsey”).
Additionally, the gene that controls the AMH type II receptor (AMHR2) is also associated with follicle recruitment, further connecting AMH to follicle recruitment (see, Voorhuis M., Broekmans F. J., Fauser B. C., Onland-Moret N. C., van der Schouw Y. T., Genes Involved in Initial Follicle Recruitment May be Associated With Age at Menopause, Journal of Clinical Endocrinology & Metabolism, March 2011, 473-479). Because of the connection of AMH to follicular recruitment and growth, AMH levels are widely considered to best reflect TOR (see, Ledger; Nelson I). Because TOR is the primary component of ovarian age, low AMH levels are indicative of ovarian aging and AMH levels below normal for a particular age are indicative of premature ovarian aging.
Because of the association of AMH with FOR and TOR, an AMH test with levels below age-specific normal levels can indicate POA. As discussed above, POA affects approximately 10% of all women, and can have different causes, including, but not limited to, the factors set forth in Table 1:
TABLE 1KNOWN CAUSES OF PREMATURE OVARIAN AGINGLow number of follicles/oocytes at birth/menarcheKnown genetic causesExcessive follicle recruitmentAnti-ovarian autoimmunityAutoimmune oophoritisAnti-ovarian autoimmunityAutoimmune polyglandular syndromesTurner syndromeSpace occupying lesionsEndometriosisOvarian tumorsIatrogenic interventionsSurgeryChemotherapyRadiation therapyBone marrow transplantationAnti-viral therapies
As Table 1 shows, aside from iatrogenic (caused by medical treatment) follicle/oocyte losses and ovarian tissue loss from space-occupying lesions, premature ovarian aging has other causes, such as excessively rapid recruitment of follicles, low follicle numbers at birth and/or menarche, genetic disorders and anti-ovarian autoimmunity. Both low follicle numbers at birth and excessively rapid recruitment are under strong genetic control. The other major causes of POA, as discussed below, are also under genetic control.
Approximately one-third of POA cases are caused by anti-ovarian autoimmunity (see, Gleicher N., Weghofer A., Oktay K., Barad D., Do Etiologies of Premature Ovarian Aging (POA) Mimic Those of Premature Ovarian Failure (POF)? Human Reproduction, October 2009, 2395-2400). Anti-ovarian autoimmunity is well-known in humans with Addison's disease who develop autoimmune (lymphocytic) oophoritis, autoimmune polyglandular syndromes (APS), and Turner's syndrome. (see, Hoek A., Schoemaker J., Drexhage H. A., Premature Ovarian Failure and Ovarian Autoimmunity, Endocrinology Review, February 1997, 107-134, referred to hereinafter as “Hoek”). Hoek also reveals that ovaries are often subject to an autoimmune attack that is statistically associated with thyroid autoimmunity, anti-adrenal autoimmunity and other, often non-organ-specific, autoimmune responses. The X chromosome's role as an autoimmune chromosome also explains the association of autoimmunity and Turner syndrome (see, Bianchi I., Lleo A., Gershwin M. E., Invernizzi P., The X Chromosome and Immune Associated Genes, Journal of Autoimmunity, May 2012, 187-192; Bukalov V. K., Gutin L., Cheng C. M., Zhou J., Sheth P., Shah K., Arepalli S., Vanderhoof V., Nelson L. M., Bondy C. A., Autoimmune Disorders in Women with Turner Syndrome and Women with Karyotypically Normal Primary Ovarian Insufficiency, Journal of Autoimmunity, June 2012, 315-322; and Lleo A., Moroni L., Caliari L., Invernizzi P., Autoimmunity and Turner's Syndrome, Autoimmune Review, May 2012, 538-543). Therefore, autoimmune attacks on the ovaries are known in the art, but their precise mechanisms are not well understood.
Autoimmune-associated premature ovarian aging is most understood in combination with autoimmune polyendocrine syndrome type 1 (APS-1), also known as polyendocrinopathy candidiasis ectodermal dystrophy or Whitaker syndrome. It is caused by a mutation in the autoimmune regulator (AIRE) gene (see, Michels). This gene is of crucial importance in the thymus, where it regulates the process that prevents T cells from attacking a human's own cells. AIRE mutations that interfere with normal AIRE activity are associated with attacks against a human's own cells. The connection between AIRE and premature ovarian aging is supported by animal models. AIRE gene knockout mice experience early follicle depletion by age 20 weeks and complete follicle depletion (POF/POI) in 50-60% of animals. Therefore, AIRE appears crucial for preventing premature ovarian aging, and mutations in the gene de-inhibit follicle maturation, leading to the rapid depletion discussed above. Because of AIRE's strong association with autoimmunity, impaired fertility in the AIRE knockout mouse model can be attributed to immune-mediated loss of TOR. Such immune-mediated loss of TOR is caused by autoimmune attacks on the ovaries, thereby destroying the oocyte reserve. The AIRE gene is the first gene associated with autoimmune-induced premature ovarian aging (see, Michels; and Cushman R. A., Evidence That the Autoimmune Regulator Gene Influences Thymic Production of Ovarian Antigens and Prevents Autoimmune-Mediated Premature Reproductive Senescence, Biology of Reproduction, April 2012, 109).
Because of the link between autoimmunity and ovarian aging, any autoimmunity in females must be considered a risk factor for premature ovarian aging. Moreover, because autoimmunity is highly familial, a patient's family history of autoimmunity is also a risk factor. This includes a familial history of repeated pregnancy loss, often the consequence of abnormal immune system activation.
In addition to familial autoimmunity, other genetic influences on ovarian aging are well demonstrated. Age at menopause is well-correlated between mothers and daughters and between pairs of sisters (see, van Asselt K. M., Kok H. S., Pearson P. L., Dubas J. S., Peeters P. H., Te Velde E. R., van Noord P. A., Heritability of Menopausal Age in Mothers and Daughters, Fertility and Sterility, November 2004, 1348-1351; and Morris D. H., Jones M. E., Schoemaker M. J., Ashworth A., Swerdlow A. J., Familial Concordance for Age at Natural Menopause; Results From the Breakthrough Generations Study, Menopause, September 2011, 956-961). Additionally, age at menarche, which is also genetically influenced, relates to risk for POA (see, Weghofer A., Kim A., Barad D. H., Gleicher N., Age at Menarche: A Predictor of Diminished Ovarian Function, Fertility and Sterility, October 2013, 1039-1043). Therefore, whether a human female's mother or sister(s) entered menopause early and/or a human female's own young age at menarche should also be considered risk factors for POA.
All of the publications mentioned above, as well as those mentioned below, are incorporated by reference herein.